The present invention concerns a human plasma catecholamine assay which employs high pressure liquid chromatography with electrochemical detection (HPLC-ECD) after prior extraction of the catecholamines from plasma using the boric acid method of Smedes et al (J. Chromatogr. 231:25-39, 1982). The novel aspects of the method include the unique combination of equipment used in the assay, the chromatography conditions and the maintenance procedures developed to sustain the assay at a high level of sensitivity over a prolonged period of time.
Catecholamines are members of a family of molecules referred to as biogenic amines which include indoleamines, their metabolites and derivatives. In animals, catecholamines (CAs) act as neurotransmitters and hormones. Molecules possessing similar structures are also found in plants but their exact function in vivo has not yet been fully elucidated. CA-like derivatives of certain plants comprise clinically important drugs (e.g. ephedrine and reserpine). It is emphasized that the procedures described herein are equally effective on all biogenic amines derived from a multiplicity of sources (including physiological fluids other than plasma, tissue extracts and synthetic materials).
The molecules which can be detected by the assay techniques described herein include sympathomimetic amines such as amphetamines and their derivatives, indoleamines and their derivatives, the belladonna alkaloids and their derivatives, neuroleptic and non-neuroleptic analgesics, certain anesthetics, many anti-histamines, anti-adrenergic drugs, xanthines, opioids (and their derivatives and antagonists), tricyclic antidepressants, tranquilizers, .beta.-blockers, and so on. In short, many nervous system-active substances and other putative neurotransmitters are functional derivatives of the catecholamine nucleus and, as such, are amenable to the analysis procedures described herein.
The following discussion will focus on the study of catecholamines (CAs) in resting human plasma as the model system because this particular analysis presents the greatest analytical challenge. The methods described herein can also be used to detect the naturally occurring metabolites and derivatives of the principal CAs. Such metabolites are found concurrently in plasma and other physiological fluids; however, because these compounds are inherently more stable and present in higher concentrations than the plasma CAs, their analysis is considerably easier to perform and will not be discussed herein. The instability and low concentration of plasma CAs have made their analysis very difficult and the reliability and accuracy of methods developed previously have been the subject of controversy. The invention described herein has overcome the earlier problems encountered in the assay of plasma CAs.
1. Introduction
One entity responsible for the maintenance of vertebrate life is the central nervous system (CNS). The CNS communicates with the body of the organism by means of the peripheral nervous system. The CNS receives information regarding both sensation and organ function along afferent pathways and it sends instructions to the body on the efferent circuits.
Transmission of information is an electrochemical process: nerves propagate signals electrically along their own length. When these signals reach their target (be it another nerve cell or a target tissue) the nerve ending releases chemical signals which diffuse to the target and combine with it, thereafter eliciting responses from it. Compounds belonging to this special group of chemicals (which nerves use to relay information) are known as neurotransmitters. The electrical nerve signal itself is known as an action potential and has a duration of about one millisecond. Each action potential releases a quantum of neurotransmitter and information is coded by the frequency and pattern of the electrical impulses.
One part of the CNS, namely the autonomic nervous system (ANS), is primarily responsible for the day-to-day maintenance of a variety of critical, physiological functions and balances in the mammalian body. The ANS is a self-regulating system. It functions by means of a balance between two neural networks which can be viewed as an "accelerator" and a "brake". That is, instructions are sent via the two neural networks in an "ON-OFF" code with the end result being an integration of the signals received (most structures are innervated by both networks). Generally speaking, "OFF" instructions are sent down parasympathetic nerves (using the neurotransmitter, acetylcholine) and "ON" instructions are sent down sympathetic nerves (using the catecholamines). The designation "catecholamine" refers to a compound composed of a catechol nucleus (a benzene ring with two adjacent hydroxyl groups) and an amine-containing side chain. [The parent compound is .beta.-phenylethylamine.] The most important CAs known to occur in humans are dopamine (DA), noradrenaline (NA) and adrenaline (A).
The parasympathetic nervous sytem is concerned with the functions of conservation and restoration of energy (i.e. vegetative aspects of day-to-day living) and is organized for discrete and localized action rather than mass response. It will slow down the heart rate (hence the "brake" analogy), lower the blood pressure, stimulate gastrointestinal movements and secretions, aid in absorption of nutrients, protect the retina from excess light and empty the urinary bladder and rectum.
The sympathetic side of the ANS is concerned with the expenditure of energy and the coordination of processes which allow the organism to deal with stress (hence the "accelerator" analogy). The most important neurotransmitter of this system is noradrenaline (or, "norepinephrine") and the network is often referred to as the adrenergic system. Among other effects, the sympathetic nervous system (SNS) accelerates the heart, raises the blood pressure, dilates the bronchioles, inhibits peristalsis of the intestines, causes the breakdown of glycogen into glucose and the liberation of free fatty acids (thereby supplying energy), and shifts blood to skeletal muscle. These actions may be significantly modified by the liberation of adrenaline (or "epinephrine") from the adrenal medulla into the blood stream. Hence, the "sympathoadrenal system" is organized in such a way so that the structures which it innervates can be orchestrated simultaneously and with great force. It is to be appreciated that, in mammals, an action potential can travel as fast as 120 meters/second along a nerve fibre whose diameter is, in turn, measured in angstroms. The nerve cell body ranges in length from 10-300 .mu.m in vertebrates, to 300-800 .mu.m in certain invertebrates, and the fibres which conduct the action potentials are no larger than 20 .mu.m in diameter in vertebrates. The sympathoadrenal system is "on" all the time but the degree of activity varies from moment to moment and from organ to organ.
It will be obvious from the preceding discussion that NA and A are intimately involved in the day-to-day maintenance of normal vertebrate life. These two CAs are also thought to play a role in many pathological processes which disrupt the function of the structures and/or organs modulated by them. In the CNS, dopamine functions as a neurotransmitter but is of interest in the periphery mainly as a precursor of NA. Therefore, quantification of NA and A would, at least potentially, provide an intimate window into the state of an organism from a sympathetic nervous system point of view. As well, periodic monitoring of NA and A would yield a dynamic assessment of that state. Thus, the importance of the quantification of NA and A as a clinical and research tool cannot be overstated.
From an experimental point of view, there are a number of possible approaches to accomplishing the task of quantifying NA and A. The neurotransmitter outflow of sympathetic nerves spills over into the various physiological fluids (including blood plasma, urine and cerebrospinal fluid). The assaying of these fluids for their catecholamine content can provide information generally not available through other techniques. For example, while it is technically possible, to a limited extent, to study the sympathetic nerves to skin and skeletal muscle by microneurographic electrophysiological methods, the nervation of internal organs is not accessible for such testing. However, the variations in sympathetic neurotransmitter activity of internal organs may be assessed by biochemical means, namely by the measurement of the CA content of physiological fluids. Each of these fluids is in a constant state of flux and dynamic changes can be monitored within each one. By so doing, it should be possible to at least infer information regarding the level of nerve activity in the organ of interest and under the conditions of study. The monitoring of blood plasma levels of CAs offers the current best solution to assessing the state and activity of certain internal organs and systems from a sympathetic nervous system point of view. However, to provide meaningful data from assays of plasma CAs, it is necessary to balance the dichotomy of physiological dynamicism and methodological limitations.
Heretofore, severe limitations have been placed on the ability to quantify the concentrations of plasma catecholamines due in part to the ethereal quality of the catecholamines themselves. They are small compounds (less than 200 molecular weight) and are inherently unstable, having a half-life in plasma of only about two minutes. Furthermore, CAs are present in extremely minute quantities, viz., picograms per milliliter of plasma. For these and other reasons, methods for quantifying CAs have been slow in development.
The assessment of catecholamines in plasma is also constrained by the dynamic nature of the human nervous system. Some of the limitations imposed thereby are, first, that the assessment (in whatever form it might assume) cannot disrupt normal function or in any way alter it (i.e. affect the sympathoadrenal system, or "accelerator"); secondly, by implication, the technique must be as non-invasive as possible and be repeatable and reproducible; thirdly, it should be as realistic as possible in terms of having a minimum of special equipment and/or handling requirements.
The following summary of the life cycle of catecholamines is given in the interest of providing a background as to the significance and interpretation of the assay techniques described thereafter.
2. Synthesis and Release of Catecholamines
Catecholamines are produced and released into physiological fluids through the following mechanism. From a geographical point of view, nerves lie in very close proximity to blood vessels throughout the body. The amino acid tyrosine leaves the blood and enters the nerve fibre terminal varicosity by a special concentrating mechanism. Tyrosine is then converted in the cytoplasm to L-Dopa by the enzyme tyrosine hydroxylase (which is found only in CA-producing cells). This reaction proceeds very slowly in vivo and is considered to be the rate-limiting step in the biosynthesis of the CAs. Tyrosine hydroxylase is inhibited by CAs and this feedback inhibition appears to be important in controlling the rate of biosynthesis of NA in the sympathetic nerves. L-Dopa is, in turn, rapidly decarboxylated to dopamine by the enzyme dopa decarboxylase. The dopamine then enters minute, granulated vesicles (400-600 .ANG. size) in the sympathetic nerve terminals where it is finally hydroxylated by dopamine-.beta.-hydroxylase (D.beta.H) to L-norepinephrine. D.beta.H is found only in cells that produce NA. The NA remains protected and inactive inside these storage vesicles until liberated by a nerve impulse. The vesicles move to the surface of the cell membrane in response to an action potential and expel their contents into the synaptic cleft at the nerve ending in a process known as exocytosis. The neurotransmitter diffuses across the gap to combine with specific receptors on the post-synaptic membrane and thereby elicits responses in the post-synaptic target (or effector) cell.
The nature of the response varies according to the type of effector cell contacted: the cell could be a vascular smooth muscle cell, myocardial cell, adipocyte, myometrial cell, hepatocyte or another neuron. The extent of the physiological response of the target cell to secreted or injected CA will depend upon (1) the actual fraction of CA delivered to the target cell, (2) the ability of the cell to inactivate the delivered CA, and (3) the sensitivity (including receptor and post-receptor properties) of the target cell to the CA. This is how the "fine-tuning" and coordination of so many diverse cell types and structures is possible to effect simultaneously.
Inactivation of the CAs can occur by several different pathways. The primary one is re-uptake back into the same nerve varicosity which only moments before had released them. Once back inside the terminal, the CA molecules are re-stored in the vesicles and thus recycled. This is a highly specific, high-affinity and very rapid process called "Uptake 1" (or, Neuronal Uptake). Under normal conditions, this pathway predominates. When very high levels of CA are released--by, for example, continuing stimulation of the adrenal medulla or intravenous injection of CA--then a significant proportion will be removed by re-uptake into non-neuronal tissues such as muscle, connective tissue, liver and kidney. This is called "Uptake 2" (or, Extra-Neuronal Uptake) and in this case, the CA molecules are catabolized by enzymes. The two principal enzymes which digest the CAs are monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). The metabolic products formed include normetanephrine, metanephrine, VMA (3-methoxy-4-hydroxy-mandelic acid), MHPG (3-methoxy-4-hydroxyphenylglycol), DOPAC (3,4-dihydroxyphenylacetic acid), HVA (homovanillic acid), DOPET (3,4-dihydroxyphenylethanol), 5-HIAA (5-hydroxyindoleacetic acid), and DOPEG (3,4-dihydroxyphenylglycol). Some of these compounds can, in turn, be conjugated to their respective sulfates and glucuronides.
Primarily because of the efficiency of these uptake mechanisms, only a small fraction (perhaps 10-20%) of the physiologically active CA released by the terminals reaches the receptors on the target cells and activates them. Considering the sweeping powers of the CAs to turn cells on and off, it is appropriate that only small amounts of these compounds are needed to mediate their effects.
3. Significance of Plasma Catecholamine Studies
As was indicated earlier, nerves lie in close proximity to blood vessels; therefore, neurotransmitter spillover will rapidly appear in the blood stream. The same is true of extracellular and cerebrospinal fluids. In accordance with the preceding discussion on the metabolism of CAs, one would also expect to find CA metabolites from neuronal and non-neuronal tissue, in turn, spilled over into these fluids. Radiotracer studies have demonstrated that this is indeed the case.
Urine, on the other hand, is essentially an ultrafiltrate of plasma and is collected over a period of several hours. Compounds in urine depict activities taking place throughout the body, including the brain. For this reason, CA metabolites (as opposed to the parent molecules) predominate in the urine and thus represent an integrated image of many events which took place in the past. Metabolites are usually more stable than the parent molecule and this is especially true in the case of CAs. Additionally, CA metabolites in urine have time to accumulate to the nanogram per mL concentration range as opposed to plasma CA levels which are in the picogram per mL range.
The choice of which physiological fluid to sample for CAs is based on the kind of information about the body that is required. If one is attempting to obtain an answer to a question regarding the function of the CNS then analysis of cerebrospinal fluid is preferred. However, the routine periodic sampling of cerebrospinal fluid (CSF) is neither ethical nor practical; therefore, this is not a solution that can be applied to the problem of the state of the SNS except under special circumstances (e.g., during neurosurgery).
If, on the other hand, one is only interested in global trends in the adrenergic system (such as the secretion of CAs during the night as an indication of an active pheochromocytoma), then urinalysis can provide an adequate assessment.
If an immediate answer as to the state of the adrenergic nervous system is required (such as prior to, and following, surgery or after a myocardial infarction), then clearly a sample of plasma should be assayed. Most often, a plasma assessment is preferred because of the dynamic nature of the SNS. In both research and clinical situations, one is trying to gain information about a system that is highly influenced by the state of the organism at that point in time and in that particular state of health. Under the background of a constantly shifting scale, it is more physiologically relevent to catch this dynamicism at the time, and under the conditions, it takes place rather than attempting to infer information about events past at an unspecified location.
However, it must be emphasized that only a very small amount of CA diffuses away from the synaptic cleft and into the circulation. The actual amount of CA escaping from any one organ or structure will be the integrated result of a number of contributing factors. These include the sympathetic nerve firing rate, the density of the sympathetic nerves in that organ or area, the mass of the organ, the width of the synaptic cleft, the capacity of neuronal and non-neuronal tissue for re-uptake and/or enzymatic degradation, the permeability of the local capillaries to the CAs, and the blood flow through the organ. Obviously, these parameters will also vary tremendously between organs and according to need over time. It is known that the half-life of NA in plasma is approximately 2 minutes; therefore, if it is desired to know what is happening within a structure (from a sympathetic nervous system point of view), blood should be sampled periodically over some unit of time (e.g., every six minutes for an hour).
It is important to realize that the CAs which escape into the bloodstream (and other physiological fluids) are not at liberty to float freely: most are in fact conjugated to sulfate molecules. Only a small proportion of the total amount of any CA (perhaps 10 to 20%) exists in plasma in the free, i.e., unbound, non-derivatized form. [In the case of dopamine, this proportion is even lower (i.e. 5%).] It is this free amount of CA that is of interest in clinical and research medicine. The exact function of the sulfoconjugated CAs is not known and the mechanisms of the equilibria which create and maintain this pool remain to be elucidated. It has, however, been demonstrated that it is the amount of free CAs which fluctuates in response to stimuli; therefore, measurement of this fraction constitutes a valid appraisal of CA dynamics.
Having established that one wishes to know the CA content of plasma, the problem remains of the choice of location(s) for such a sample. Logically, it would have to be from the venous outflow of the organ (or system) being studied. This requires that the assumption be made that no NA came into the organ (on the inlet, or arterial side) and that the organ did not extract any NA that may have been present there. These may or may not be safe assumptions. In any event, selective catheterization of individual organs is not practical. What is practical is a venous blood sample from a peripheral vein. Because many organs are necessarily spilling excess CAs into the blood from a wide range of physiological tasks, it would seem that such a blood sample would not provide any useful information. Fortunately, it does. The venous blood levels of NA and A do parallel the known level of sympathetic tone in a number of clinical and experimental situations [Ref.: Esler, Hasking et al, J. Hypertension 3:117-129, 1985.]. Even though what is being sampled is some net result of events occurring throughout the body (including the brain) the blood levels of NA and A do fluctuate in a coherent fashion. For example, at rest, the normal plasma level (blood being part cells and part fluid, i.e., plasma) of NA is about 150-300 pg/mL (picograms per milliliter) and A is about 25-75 pg/mL. However, during exercise, these values can climb to 5,000 pg/mL NA and 1,500 pg/mL A.
Occasionally, it has been reported that a patient with a tumor of the adrenal medulla, known as a pheochromocytoma, will have plasma levels of NA of 10,000 pg/mL and A of 4,000 pg/mL. This latter amount of epinephrine is essentially lethal, as is the tumor if allowed to grow unchecked. Since these changes from resting values are at the level of orders of magnitude, they would not often be confused with normal physiological concentrations of CAs. Therefore, it is safe to conclude that in most settings by measuring plasma levels of CAs in peripheral blood, a useful index of SNS activity is obtained. Only under special circumstances is it possible to study regional sympathetic response patterns, as represented by CA overflow, through sampling of blood from selected vessels.
It should be borne in mind that epinephrine is mainly synthesized as a hormone in the adrenal medulla. Therefore, any increase in the concentration of epinephrine in plasma will indicate increased adreno-medullary secretion. Norepinephrine, on the other hand, is primarily a neurotransmitter released by post-ganglionic sympathetic nerve endings. At low nerve impulse rates, about 90% of the norepinephrine released by the nerve terminal is removed by re-uptake (Uptake 1) back into the same nerve terminal. It is only at high nerve impulse rates (or inadequacy of the re-uptake system) that higher amounts of norepinephrine can escape into the bloodstream. It follows that this is the explanation for the low, resting concentrations of norepinephrine and epinephrine that are normally encountered. Thus plasma CA measurements will be a more reliable index, or accurate indicator, of adrenergic activity when the adrenergic stimulus is more intense.
The measurement of plasma levels of CAs is always accompanied by the simultaneous collection of other physiological data such as heart rate, blood pressure, oxygen consumption and the like. Taken in isolation, CA concentrations are usually relatively meaningless. Furthermore, it is vital that in any study of sympathetic nervous system function, individual subjects act as their own controls because CA responses are so individually tailored to the needs of a single organism.
The exact factors responsible for the maintenance of the dynamic equilibrium of CA levels in plasma, their mechanisms of action and the magnitude of their effects on plasma CA levels is a separate issue from the fact that CAs appear in plasma at all. It is precisely because of the methodological limitations which have heretofore existed in this field that the amount of actual (as opposed to apparent) information on CA dynamics is scant. Controversies persist because definitive studies cannot be performed without standardized analytical techniques which are available to laboratories in many centres with different research foci. [Ref.: Hjemdahl, Acta Physiol. Scand. Suppl., 527: 43-54, 1984.] For these reasons, the full characterization of CA metabolism and function remains to be performed.
Finally, it must be borne in mind that the assay, as described herein, will refer only to analysis of plasma samples obtained from subjects who are drug-free. That is, they have not taken drugs (defined as any foreign substances) which would either interfere with the functioning of the body in vivo (i.e. the synthesis, release and metabolism of the CAs) or with the performance of the assay (e.g., the separation of the peaks of interest on the chromatogram).
4. Summary of Catecholamine Assay Techniques
The discussion hereinafter will be concerned with the elucidation of the levels of CAs found in human plasma samples. This model (i.e., human plasma CAs) is used because this particular analysis presents the greatest analytical challenge. It is stressed that structurally similar compounds, their derivatives and metabolites, obtained from other sources, can also be assayed utilizing the methods described herein but such compounds and their assay will not be described.
Each one of the catecholamines (i.e., A, NA and DA) has different physiological and physicochemical properties. This latter characteristic has been exploited to develop assay techniques. Historically, these assays have proven to be a challenge both from the point of view of the compounds being tested and from the point of view of the technology available.
As mentioned above, catecholamines are small molecules (less than 200 molecular weight) with very subtle structural differences; they are normally present in very low quantities in plasma (picograms of the native CAs in plasma versus nanograms of stable metabolites in urine or nanograms of CAs in tissue) and have a very short half-life (less than two minutes) in plasma. Furthermore, CAs are unstable molecules which decompose in alkaline solutions, upon exposure to light, and even if brought to temperatures greater than +5.degree. C. Obviously, before plasma CAs can be measured, they must first be stabilized, separated from the other constituents of plasma and from each other, and finally, they must be "visualized" in some quantifiable fashion. Whatever methodology is chosen to achieve this end must also be reproducible and reliable.
Stabilization of the CAs in a blood sample is most easily achieved by collecting the blood into a chilled, silicone-lined tube; the blood sample should be placed on ice and in the dark as soon as it has been collected. It is important that the plasma be separated from the cellular fraction of blood as soon as feasible because red blood cells contain catabolic enzymes such as catechol-o-methyltransferase. Depending on the assay technique to be used, (a) chemical stabilizer(s) may be added to the plasma. If it cannot be assayed immediately, the plasma sample must be frozen at -70.degree. C. or lower.
Having stabilized the plasma, the CAs must then be separated away from the other constituents of the plasma. Blood plasma is a complex matrix comprising thousands of molecules of many different chemical types, molecular sizes and structures. In the case of the unstable CA molecules, it is essential that the extraction and quantification processes be as brief and as accurate as possible. A plethora of methods have been tried in an attempt to divorce the CAs away from the other constituents of plasma or else to quantify them, in situ. as expeditiously as is practical. These methods have achieved varying degrees of success and will be reviewed hereinbelow in terms of the availability of new technologies so as to put the present invention in context with this field of analysis.
Prior to the mid-1950's, the only way to find out whether or not plasma contained any CAs was to use a bioassay. In these techniques, crude extracts of plasma were injected into a small mammal (e.g., a rabbit) and heart rate was monitored. While these tests were sensitive, they gave little specific information regarding the actual amount or type of CA present. Furthermore, reproducibility depended entirely on the skill of the operator and was not transferable.
Since the 1950's, there have been three major revolutions in available technology and all have resulted in improved CA assays. These methods represent attempts to improve the specificity and sensitivity of CA measurements while reducing the amount of interference by other compounds. They are:
(A) Development of the fluorometric assay (mid-1950's to mid-1960's); PA1 (B) Development of the radioenzymatic assay (mid 1960's to mid-1970's); PA1 (C) Development of an assay utilizing high pressure liquid chromatography with electrochemical detection (HPLC-ECD) (late 1970's to present). PA1 (a) Solvent (mobile phase) reservoir(s) PA1 (b) Pump PA1 (c) Injector (i.e. a means by which to introduce the sample to be analyzed into the system) PA1 (d) Column (containing the stationary phase) PA1 (e) Detector PA1 (f) Data recorder(s)
Other methods of separation and detection of CAs have been tried, including Gas Chromatography-Mass Spectrometry (GCMS), radioimmunoassays and a variety of techniques employing detection of the CAs by electron-capture detectors, ultraviolet absorption and fluorescent spectroscopy but none of these has, to date, proven to be of practical value. [Ref.: Holly and Makin, Anal. Biochem. 128(2):257-274, 1983.]
The use of monoclonal antibodies for the isolation of CAs from plasma has so far been unsuccessful due to the lack of specificity of the antibodies produced. Recently, the CA receptor was successfully cloned and it is hoped that this product will eventually be made available for CA assays. However, any resulting specific assay technique(s) remain(s) to be developed. Therefore, the three assay methods in the above list represent the practical advances, to date, for the quantification of CAs.
To put the significance of the present invention (an HPLC-ECD plasma CA assay) into perspective, the following is a short review of the fluorometric and radioenzymatic assay methods.
(A) Fluorometric Assay
The fluorometric assay is based on the fact that CA molecules possess natural fluorescence. The CAs and their metabolites have characteristic emission spectra which can be exploited, to some extent, to differentiate between them. The idea behind an assay based on this fact was to be able to detect levels of CAs in physiological samples with a minimum of pretreatment (i.e. isolation from plasma). Normally, the plasma was only deproteinized prior to analysis. However, not only is the native fluorescence of the CAs very weak and non-specific, but these molecules are present in such low concentrations in plasma that they can barely be detected by available instruments. This limitation can be overcome by the derivatization of the CAs to larger molecules which have strong native fluorescence (for example, trihydroxyindoles and o-phthalaldehyde have been used) thereby rendering the CAs more visible and facilitating their detection.
One of the major advantages of this method is that it is quick to perform and comprises relatively few steps; additionally, the equipment required is inexpensive and readily available. Among the major disadvantages of this type of assay is the complication that plasma contains many compounds (other than those of interest) and some of these are susceptible to the isolation and concentration procedures employed. Such compounds may also fluoresce, thereby invalidating the assay. Many common foods, such as bananas, chocolate and coffee, and frequently prescribed drugs, such as antibiotics, fall into this category.
Furthermore, even with derivatization, the concentrations of CAs in resting plasma are so low as to be undetectable. An additional problem with the derivatization is that the molecules thus created are very unstable and must be constructed and quantified under strictly controlled conditions or else they will, literally, disappear from the detection system. It is also not possible to differentiate between the CAs (DA, NA, A); therefore, the assay can only quantitate the total amount of CAs present rather than characterize their individual concentrations. In time, the assistance of alumina extraction, isotope-labelling and chromatographic separation were added to the fluorometric procedure but neither the specificity nor the sensitivity of this assay method could be significantly improved without heroic efforts. All of these restrictions also have implications for the reproducibility of the method, which is limited. These limitations have precluded the utility of this method to measurements of plasma CA levels. Therefore, this assay is largely reserved for the analysis of urine samples which contain nanogram quantities of CA metabolites (i.e. 1.times.10.sup.-9 grams of stable metabolites).
Hospital laboratories often maintain a fluorometric assay for total urinary CAs to use as a screen for the above-mentioned type of life-threatening tumor of the adrenal medulla (pheochromocytoma). This tumor secretes extremely high levels of CAs thereby facilitating the diagnosis. In this instance, information regarding urinary levels of CA metabolites can be very useful.
The recent introduction of laser technology to the field of fluorescence detection is expected to improve the sensitivity of this assay method (see below). However, the problems of the stability of the analate, specificity, sensitivity, and reproducibility of the fluorometric assay must still be overcome.
(B) Radioenzymatic Assay (REA) of Plasma Catecholamines
Clearly, the fluorometric assay does not constitute a resolution of the problems of effectively extracting, separating and detecting the individual CAs of human plasma. The next major attempt at solving these problems entailed the enzymatic conversion of the CAs to their more stable metabolites. It could be arranged that the metabolites thus formed were radiolabelled thereby simplifying quantification once the molecules had been separated from the plasma and from each other. This separation was effected by the use of chromatography. Until the advent of HPLC-ECD technology, the radioenzymatic assay method was the most useful means by which to quantify plasma CAs. It is instructive to subsequent discussions herein to clarify the basic principles of chromatography before the details of this assay are reviewed. (Both the radioenzymatic and HPLC-ECD catecholamine assays utilize chromatography.)